Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome

Kindler syndrome is an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Linkage and homozygosity analysis in an isolated Panamanian cohort and in additional inbred families mapped the gene to 20p12.3. Loss-of-function mutations were identified in the FLJ20116 gene (renamed “KIND1” [encoding kindlin-1]). Kindlin-1 is a human homolog of the Caenorhabditis elegans protein UNC-112, a membrane-associated structural/signaling protein that has been implicated in linking the actin cytoskeleton to the extracellular matrix (ECM). Thus, Kindler syndrome is, to our knowledge, the first skin fragility disorder caused by a defect in actin-ECM linkage, rather than keratin-ECM linkage.     

Influence of cultivation conditions on citrate production by Aspergillus niger in a semi-pilot-scale plant

The influence of some fermentation parameters on the semi-pilot scale (alteration of growth conditions,e.g., sugar concentration, incubation temperature and initial pH) on citrate production was demonstrated in parent and mutant strains ofAspergillus niger. Raw material from sugar industry (cane molasses) was examined as basal fermentation medium in a stirred stainless-steel 15-L fermentor. After growth on medium with 150 g/L sugar, the parent strain produced 51.2 g/L citric acid; the mutant strain achieved production maximum of 96.2 g/L. Comparing the growth, kinetic (volumetric substrate uptake rate, rate of substrate consumption and volumetric productivity rate) and production parameters it was found that the mutant strain grows more rapidly, with slightly changed morphology (intermediate, shiny round pellets with diameter 0.6–0.7 mm), and exhibits a higher citrate production and higher efficiency of sugar utilization.     

The application of confocal microscopy to the study of liposome adsorption onto bacterial biofilms

Confocal laser scanning microscopy has been used to visualise the adsorption of fluorescently labelled liposomes on immobilised biofilms of the bacterium Staphylococcus aureus. The liposomes were prepared with a wide range of compositions with phosphatidylcholines as the predominant lipids using the extrusion technique. They had weight average diameters of 125 +/- 5 nm and were prepared with encapsulated carboxyfluorescein. Cationic liposomes were prepared by incorporating dimethyldioctadecylammonium bromide (DDAB) or 3, beta [N-(N1,N1 dimethylammonium ethane)-carbamoyl] cholesterol (DC-chol) and anionic liposomes were prepared by incorporation of phosphatidylinositol (PI). Pegylated cationic liposomes were prepared by incorporation of DDAB and 1,2-dipalmitoylphosphatidylethanolamine-N-[polyethylene glycol)-2000]. Confocal laser scanned images showed the preferential adsorption of the fluorescent cationic liposomes at the biofilm-bulk phase interface which on quantitation gave fluorescent peaks at the interface when scanned perpendicular (z-direction) to the biofilm surface (x-y plane). The biofilm fluorescence enhancement (BFE) at the interface was examined as a function of liposomal lipid concentration and liposome composition. Studies of the extent of pegylation of the cationic liposomes incorporating DDAB, on adsorption at the biofilm-bulk phase interface were made. The results demonstrated that pegylation inhibited adsorption to the bacterial biofilms as seen by the decline in the peak of fluorescence as the mole% DPPE-PEG-2000 was increased in a range from 0 to 9 mole%. The results indicate that confocal laser scanning microscopy is a useful technique for the study of liposome adsorption to bacterial biofilms and complements the method based on the use of radiolabelled liposomes.     

Transition metal acetylsalicylates and their anti-inflammatory activity

Mononuclear and binuclear transition metal [Co(II), Cu(II), Ni(II) and Zn(II)] acetylsalicylates of the type [M(L)2], [M(L)2Cl2] and [(M)2(L)4] have been prepared and characterized on the basis of their physical, spectral and analytical data. The complexes have been investigated in an in vivo animal model for anti-inflammatory activity and show a better effect and a more potent action than acetylsalicylic acid.     

Oesophageal cancer in Africa

Oesophageal cancer, the eighth most frequent cancer in the world occurs as two main subtypes, squamous cell carcinoma (more prevalent in developing countries) and adenocarcinoma (more common in developed countries). Certain populations of central, eastern, and southern Africa display very high frequencies of oesophageal squamous cell carcinoma, presenting a serious health burden to the continent. Most patients are diagnosed at a late stage because of the asymptomatic development of the disease, with associated poor prognosis. A better understanding of the aetiological agents and molecular mechanisms involved in the development of oesophageal squamous cell carcinoma may offer opportunities to reduce exposure to environmental risk factors and also allow early diagnosis or predict response to therapy. Epidemiologic studies have identified smoking, alcohol consumption, diets poor in fresh fruit and vegetables, consumption of foods contaminated with Fusarium verticillioides, and HPV infection as risk factors associated with the development of this disease in Africa. Although we have an incomplete understanding of the molecular events involved in the development of oesophageal squamous cell carcinoma, advances have been made that suggest lines of future exploration. South African patients with oesophageal squamous cell carcinoma display a lower incidence of point mutations in the p53 gene than described elsewhere, suggesting that the profile of aetiological agents may be different than described for other high-risk areas for oesophageal cancer. Recent studies suggest that RARbeta and COX II is frequently downregulated and upregulated, respectively, in oesophageal squamous cell carcinomas. These results suggest potential therapeutic opportunities that can be exploited to combat the high incidence of this disease in Africa.     

Sensitive liquid chromatographic method using fluorescence detection for the determination of estradiol 3- and 17-glucuronides in rat and human liver microsomal incubations: formation kinetics

We have developed a sensitive and specific HPLC-fluorescence assay for the determination of estradiol-3-glucuronide and estradiol-17-glucuronide in human and rat liver microsomal incubations. The method utilizes a mobile phase comprised of acetonitrile and 50 mM ammonium phosphate buffer (35:65, v/v) that is pumped though a phenyl column at 1 ml/min; the run time is less than 15 min. Calibration curves for both metabolites were linear over the range 20-4000 pmol. The intra- and inter-day coefficients of variation were <6%. In both rat and human liver microsomes, the formation of estradiol-3-glucuronide displayed atypical kinetics (consistent with activation), while estradiol-17-glucuronide formation was consistent with classical Michaelis-Menten kinetics. Overall, the assay described is a sensitive and reproducible method for the determination of estradiol glucuronides in liver microsomal preparations.     

Heart and breast muscle mitochondrial dysfunction in pulmonary hypertension syndrome in broilers (Gallus domesticus)

This study was conducted to determine function and defects in electron transport in muscle mitochondria of meat chickens (broilers) with pulmonary hypertension syndrome (PHS). The respiratory control ratio (RCR, indicative of respiratory chain coupling) was higher in the control than in PHS breast and heart muscle mitochondria, but there were no differences in the ADP/O (an index of oxidative phosphorylation). Sequential additions of ADP improved the RCR in the control breast muscle mitochondria and the ADP/O in PHS breast and heart muscle mitochondria. Basal hydrogen peroxide production, (an indicator of electron leak), was higher in PHS breast and heart muscle mitochondria than in controls and differences in electron leak in PHS mitochondria were magnified by inhibiting electron transport at Complex I and III (cyt b(562)). Complex I activity was lower in PHS heart mitochondria but there was no difference in Complex II activity. Thus, compared to controls, PHS mitochondria exhibited site-specific defects in electron transport within Complex I and III that could contribute to lower respiratory chain coupling. Additionally, it appears that healthy broilers may exhibit higher basal levels of electron leak compared to other avian species. Together, these findings provide insight into inefficient cellular use of oxygen that may contribute to the development of PHS in broilers.     

Reactive oxygen species upregulate expression of PAI-1 in endothelial cells

Second messengers involved in the signal transduction pathway leading to induction of the plasminogen activator inhibitor (PAI-1) have not yet been well characterized. This study focuses on the mechanisms of regulation of PAI-1 expression by reactive oxygen species (ROS) in human endothelial cells. Inhibition of the tumor necrosis factor alpha (TNFalpha?-induced expression of PAI-1 by antioxidant N-acetyl-L-cysteine (NAC) indicated redox-sensitive mechanisms involved in the signaling pathway. Because TNFalpha induces PAI-1 production in endothelial cells, and NAC attenuated this response, we attempted to investigate the possible involvement of ROS in the activation of PAI-1 by TNFalpha. Upregulation of PAI-1 expression in endothelial cells by the stimulation with TNFalpha (50 ng/ml) or H2O2 (10-200 micro M), observed by measurement of the antigen and mRNA levels, was reversed in the presence of NAC (20mM). The stimulatory effect of ROS was detected also at the level of the PAI-1 promoter in endothelial cells transfected with plasmid p800 LUC containing a PAI-1 promoter fragment (+71 to -800). The PAI-1 promoter activity was increased in the presence of ROS, and was suppressed by up to 75% in the presence of antioxidants. On the basis of this study we can conclude that reactive oxygen species play an important role in a cytokine-induced activation of PAI-1 expression, and may act as a signal transduction messenger.     

Study of the biological effects and DNA damage exerted by a new dipalladium-Hmtpo complex on human cancer cells

The new dipalladium complex [Pd(2)(mu-mtpo-N(3),N(4))(2)(phen)(2)](NO(3))(2) (where phen=1,10-phenantroline; Hmtpo=5,7-dihydro-7-oxo-5-methyl[1,2,4]triazolopyrimidine), (Pd(2)-Hmtpo, or complex I), interacts effectively with DNA plasmid (pBS), as studied by circular dichroism spectroscopy (CD), causing large helix distortions, altering the direction of the main DNA helix axis and producing unwinding of the DNA double helix. DNA damage induced by complex I was highly significant at 2.81 microM (ovarian carcinoma TG cell line), as assessed by comet assay, a dose at which all treated nuclei showed more than 30% DNA migration to the comet tail. DNA damage effect is a consequence of genotoxicity and not a false positive response caused by cytotoxicity. In vitro cytotoxic assay on the two human tumor cell lines TG and BT-20 (breast carcinoma), shows that doses of 0.47, 1.41 and 2.81 microM produce significant antiproliferative effects after 4 days of treatment compared with control. Complex I was highly cytotoxic at 2.81 microM causing an inhibition of viable cells of 65.5%. Cisplatin (cis-DDP) exhibits lower cytotoxic activity in TG cells than dipalladium complex (a cisplatin dose of 6.67 microM inhibits 30.3%) and does not cause migration of DNA to comet tail.